Shotgun metagenomic analysis of microbial communities from the Loxahatchee nature preserve in the Florida Everglades

Abstract

Background: Currently, much is unknown about the taxonomic diversity and the mechanisms of methane metabolism in the Florida Everglades ecosystem. The Loxahatchee National Wildlife Refuge is a section of the Florida Everglades that is almost entirely unstudied in regard to taxonomic profiling. This short report analyzes the metagenome of soil samples from this Refuge to investigate the predominant taxa, as well as the abundance of genes involved in environmentally significant metabolic pathways related to methane production (nitrogen fixation and dissimilatory sulfite reduction).

Methods: Shotgun metagenomic sequencing using the Illumina platform was performed on 17 soil samples from four different sites within the Loxahatchee National Wildlife Refuge, and underwent quality control, assembly, and annotation. The soil from each sample was tested for water content and concentrations of organic carbon and nitrogen.

Results: The three most common phyla of bacteria for every site were Actinobacteria, Acidobacteria, and Proteobacteria; however, there was variation in relative phylum composition. The most common phylum of Archaea was Euryarchaeota for all sites. Alpha and beta diversity analyses indicated significant congruity in taxonomic diversity in most samples from Sites 1, 3, and 4 and negligible congruity between Site 2 and the other sites. Shotgun metagenomic sequencing revealed the presence of biogeochemical biomarkers of particular interest (e.g., mrcA, nifH, and dsrB) within the samples. The normalized abundances of mcrA, nifH, and dsrB exhibited a positive correlation with nitrogen concentration and water content, and a negative correlation with organic carbon concentration.

Conclusion: This Everglades soil metagenomic study allowed examination of wetlands biological processes and showed expected correlations between measured organic constituents and prokaryotic gene frequency. Additionally, the taxonomic profile generated gives a basis for the diversity of prokaryotic microbial life throughout the Everglades.

Keywords: Everglades; Methane production; Nitrogen fixation; Prokaryotes; Shotgun metagenomics; Soil metagenome.

Fig. 1

Sampling sites and collection methods. a Map of locations of sampling sites along trail in the Loxahatchee National Wildlife Refuge. b PVC pipe “X” configuration used during sample collection. c Method of sample packaging and storage during collection. d Environmental conditions present at Site 1

Fig. 2

Phylogenetic and functional profile for the 17 Loxahatchee soil metagenomes. Samples are denoted by the last five digits of the IMG Genome ID, with the underscore designating the sampling sites 1–4. a Stacked bar charts represent relative phylum-level abundances for the most abundant phyla based on the taxonomic affiliation of the annotated proteins within each metagenome. b Cluster analysis of COG categories based on the relative abundances of the protein dataset within each metagenome. Heatmap is scaled by relative abundances for each row ranging from low relative abundance (blue) to high relative abundance (yellow). COG categories are as follows: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation and biogenesis; K, transcription; L, replication, recombination, and repair; M, cell wall/membrane/envelope; N, cell motility; O, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking and secretion; V, defense mechanisms; W, extracellular structures; X, Mobilome: prophages, transposons; and Z, cytoskeleton

Fig. 3

Community richness (a), evenness (b), and diversity as measured by Shannon’s and Simpson’s diversity indices (c and d) across four sites within the Loxahatchee National Wildlife Refuge